Keto-Functionalized Polymer Scaffolds as Versatile Precursors to

Dec 13, 2012 - A new methacrylate monomer with a reactive ketone side chain, ... Mechanochemical Post-Polymerization Modification: Solvent-Free Solid-...
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Keto-Functionalized Polymer Scaffolds as Versatile Precursors to Polymer Side-Chain Conjugates Jingquan Liu,†,‡ Ronald C. Li,† Gregory J. Sand,† Volga Bulmus,‡ Thomas P. Davis,*,‡ and Heather D. Maynard*,† †

Department of Chemistry and Biochemistry & California Nanosystems Institute, University of California, Los Angeles, Los Angeles, California 90095-1569, United States ‡ Centre for Advanced Macromolecular Design, School of Chemical Sciences and Engineering, The University of New South Wales, NSW 2052, Australia S Supporting Information *

ABSTRACT: A new methacrylate monomer with a reactive ketone side chain, 2-(4-oxopentanoate)ethyl methacrylate (PAEMA), was synthesized and subsequently polymerized by reversible addition−fragmentation chain transfer (RAFT) polymerization to give a polymer with a narrow molecular weight distribution (PDI = 1.25). The polymer was chain extended with poly(ethylene glycol methyl ether methacrylate) (PEGMA) to yield a block copolymer. Aminooxy-containing small molecules and oligoethylene glycol were conjugated to the ketone functionality of the side chains in high yields. Cytotoxicity of the oxime-linked tetra(ethylene glycol) polymer to mouse fibroblast cells was investigated; the polymer was found to be noncytotoxic up to 1 mg/mL. The ease with which this polymer is functionalized suggests that it may be useful in forming tailored polymeric medicines.

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INTRODUCTION Polymers with side-chain groups that can be modified easily and in high yield are important synthetic building blocks.1−3 For example, reactive polymer scaffolds provide a starting point to produce side-chain functionalized polymer therapeutics.4−6 For many applications, postpolymerization modification avoids the need to determine new polymerization conditions for each unique monomer, yet at the same time allowing comparison of a series of macromolecules without complications due to different molecular weight backbones. Ideally, the modification reactions are highly efficient and occur without the use of harsh reaction conditions and reagents. This allows for conjugation of molecules that contain sensitive groups. “Click” reactions are particularly well suited for modification of polymer chains. “Click” was first used by Sharpless to describe reactions that are highly efficient without side products.7 The quintessential example of such a reaction is Huisgen 1,3-dipolar cycloaddition.8 Typically this reaction is catalyzed by copper, yet copper-less forms have been described when the alkyne is strained.9 Other examples include thiol−ene and hetero-Diels−Alder reactions.7,10 Reaction of an aldehyde or ketone with an O-hydroxylamine is another highly efficient reaction.11,12 Although a side product is released, that product is benign (water). Other advantages of oxime chemistry are that ambient temperatures are required, the bond can be formed in water, is reversible under certain conditions, and no other reagents are necessary for the chemistry to occur. © XXXX American Chemical Society

For many applications of reactive polymers, narrow molecular weight distributions of the starting polymer scaffold are desired.4−6 For example, for use in therapeutics, broad weight distributions may result in varying toxicity or pharmacokinetics. To synthesize well-defined polymers containing reactive side chains, controlled/“living” radical polymerizations (CRPs) have been utilized such as atom transfer radical polymerizations (ATRP),5,13−23 nitroxide-mediated living free radical polymerization (NMP),24−28 and reversible additionfragmentation chain transfer (RAFT) polymerization.29−41 These polymerization techniques not only give low polydispersity indices (PDIs) but also enable targeting of specific molecular weights. CRP techniques have been employed to prepare side chain polymers that could be used in oxime “click” reactions. Aminooxy side-chain polymers have been synthesized by CRP.42 Polymers containing pendent acetals that were deprotected by mildly acidic conditions to produce aldehydes were prepared both by ATRP and RAFT.23,36,43−45 Polymers synthesized by RAFT with ketal (dioxolane) side chains, which were deprotected to diols and oxidized to aldehydes, were also described.44 Examples of direct polymerization of aldehydecontaining monomers by RAFT has been demonstrated.41,46−49 The use of a ketone-containing monomer, however, not only Received: October 18, 2012 Revised: November 29, 2012

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conditions. The monomer conversions were determined by 1H NMR. The pure copolymer was obtained by precipitation into diethyl ether and methylene chloride, followed by lyophilization. 1H NMR (400 MHz, DMSO-d6) δ: 4.21, 4.09, 3.51−3.25 (overlap with H2O), 2.74, 2.13, 1.95−1.35, 0.96, 0.78 ppm. Mn(DMF) = 30 000 Da, PDI = 1.32. Conjugation of O-Benzylhydroxyamine (BHA) to pPAEMA. To a mixture of pPAEMA (12 mg, 7.64 × 10−4 mmol, Mn 15 700 Da, PDI 1.25) in 1 mL of CDCl3 were added triethylamine (5.3 mg, 5.3 × 10−2 mmol) followed by BHA hydrochloride salt (8.4 mg, 5.3 × 10−2 mmol). BHA hydrochloride slowly dissolved. The resulting mixture was kept stirring for 3 h. The CDCl3 was removed. To the resulting solid was added 10 mL of CH2Cl2, which was washed with saturated NH4Cl aqueous solution (pH 5.0) (2 × 10 mL). The organic layer was dried with Na2SO4 and evaporation gave the product. 1H NMR (400 MHz, DMSO-d6) δ: 7.26 (BHA conjugate), 4.93 (BHA conjugate), 4.09−4.00, 2.18, 1.73, 1.23−0.75. Conjugation of Carboxymethoxyamine Hemihydrochloride (CMA) to pPAEMA. To a mixture of pPAEMA (11.3 mg, 7.2 × 10−4 mmol, Mn 15 700 Da, PDI 1.25) in CDCl3/CD3OH (1:1, 2 mL) were added CMA hemihydrochloride (5.4 mg, 2.5 × 10−2 mmol) and TEA (2.5 mg, 2.5 × 10−2 mmol). The resulting mixture was stirred for 3 h. By 1H NMR, all of the ketone groups on polymer were converted into oxime bonds. The conjugate was purified by the dialysis using 6000− 8000 dialysis membrane (molecular weight cutoff MWCO: 6000− 8000 Da) in CH3OH for 24 h before drying to give the product. 1H NMR (500 MHz, DMSO-d6) δ: 4.5 (CMA conjugate), 4.35, 4.24, 2.71, 2.64, 2.55, 1.95, 1.07, 0.89 ppm. Conjugation of O-Hydroxylamine Tetra(ethylene glycol) to pPAEMA. To a solution of pPAEMA (50 mg, 3.2 × 10−3 mmol, Mn 15 700 Da, PDI 1.25) in methanol/DCM (1:1, 2 mL) were added Ohydroxylamine tetra(ethylene glycol) (TEG) (0.5 g, 2.24 mmol) and NaOAc (0.360 g, 4.39 mmol). The reaction was allowed to stir for 24 h. The solvent was then evaporated, and the residue was redissolved in deionized water. The aqueous solution was purified using centrifugation filtration (MWCO: 10 000 Da) for 10 min times 4 cycles. The filtrate was lyophilized to yield a clear oil. 1H NMR (500 MHz, CDCl3) δ: 4.26, 4.13, 3.65, 3.56, 3.38, 2.58, 2.50, 2.02−1.62, 1.01−0.86 ppm. Analysis of TEG-pPAEMA Cytotoxicity to Mouse Fibroblast Cells. A 10 mg/mL stock solution of polymer in 10% calf serum in Dulbecco’s modified eagle medium (DMEM) was prepared. This stock solution was diluted to make concentrations of 1.0, 0.1, and 0.01 mg/mL TEG-pPAEMA polymer in 10% calf serum in DMEM. Mouse fibroblast cells (NIH 3T3, ATCC) were allowed to adhere to 96 well plates prior to adding 100 μL per well of the polymer solutions. For each concentration, five replicates were used and the control was repeated 10 times. The fibroblasts were incubated with various polymer concentrations at 37 °C, 5% CO2 for 24 h. Upon removal from the incubator, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide (MTT) solutions were added according to the manufactures protocol. The supernatant was then aspirated from each well, followed by the addition of 50 μL of DMSO to dissolve the purple crystals. UV analysis at 570 nm was undertaken. The results were divided by that of the control to determine the percent viability.

avoids deprotection steps but also is more stable and less likely to oxidize than aldehydes. Vinyl ketone monomers have been polymerized by RAFT and NMP.50,51 In this report 2-(4oxopentanoate)ethyl methacrylate (PAEMA) was polymerized by RAFT polymerization. This results in polymers that contain a spacer between the ketone and backbone moiety, which could be important for conjugation reactions, particularly of bulky moieties. Synthesis and characterization of the polymers, conjugation of aminooxy compounds, and initial toxicity studies are described.

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EXPERIMENTAL SECTION

Materials. Levulinic acid (>97%, Fluka), 2-hydroxyethyl methacrylate (HEMA) (Acros, 98%), N,N′-dicyclohexylcarbodiimide (DCC) (Fluka, 99.0%), 4-(dimethylamino)pyridine (DMAP) (Fluka, 99.0%), 2,2′-azobis(isobutyronitrile) (AIBN) (Aldrich, 98%), dioxane (Fisher, 99.9%), ammonium chloride (Aldrich, 99%), O-benzylhydroxyamine hydrochloride (BHA) (Aldrich, 99%), carboxymethoxyamine hemihydrochloride (CMA) (Aldrich, 98%), triethylamine (TEA) (Acros, 99%), and poly(ethylene glycol) methyl ether methacrylate (PEGMA) (Aldrich) were used as received. The RAFT agent, 2-(ethoxycarbonyl)prop-2-yl dithiobenzoate (ECPDB) was synthesized according to a literature procedure.52 O-Hydroxylamine tetra(ethylene glycol) (TEG) was synthesized as described in the literature.53 Methods. 1H and 13C NMR spectra were acquired on an Avance DRX 400 or 500 MHz spectrometer. To obtain gel permeation chromatography (GPC) data, a Shimadzu LC-10ATvp pump was used with eluents 0.1 M LiBr in DMF (40 °C) or THF (25 °C) at a rate of 0.8 mL/min through two Polymer Laboratories 5 μm mixed-D columns (with guard column) and a RID-10A detector. The standards used for DMF and THF GPC were PMMA or PSt and PMMA, respectively. Infrared absorption spectra were recorded using a PerkinElmer FT-IR equipped with an ATR accessory. Synthesis of 2-(4-Oxopentanoate)Ethyl Methacrylate (PAEMA). Levulinic acid (2.81 g, 24.2 mmol), HEMA (3 g, 23 mmol), DCC (5.23 g, 25.3 mmol), and DMAP (0.14 g, 1.15 mmol) were mixed and stirred in DMF for 15 h. The solid precipitate was removed by filtration, and the volatiles removed by evaporation. The residue was purified by silica gel column chromatography using ethyl acetate/hexanes (2/3) as the eluent to give 5.2 g (95% yield) of a pure, colorless oil. 1H NMR (400 MHz, CDCl3) δ: 6.06 (m, 1H, CH2C), 5.52 (m, 1H, CH2C), 4.27 (m, 4H, 2 × CH2O), 2.66−2.70 (t, 2H, CH2−CO), 2.52−2.54(t, 2H, OOC−CH2), 2.10 (S, 3H, CH3−CO), 1.88 (m, 3H, CH3−CCH2). 13C NMR (100 MHz, CDCl3) δ: 206.23 (CO), 172.69 (CH2−CO−O), 166.87 (CO−CCH2), 136.10 (CCH2), 126.12 (CCH2), 62.09 (CH2O), 37.97 (H2C− CO), 29.93 (H3C−CO), 27.99 (H2C−COO), 18.29 (H3C−CCH2). RAFT Homopolymerization of PAEMA. PAEMA (1 g, 4.38 × 10−3 mol), ECPDB (39 mg, 1.45 × 10−4 mol), and AIBN (4.8 mg, 2.9 × 10−5 mol) were dissolved in DMF (2 mL). The solution was deoxygenated for 30 min by bubbling with argon and then immersed in a 70 °C bath. At 0.5, 1, 1.5, 2, and 3 h, samples were syringed from the incubating mixture under air-free conditions. The monomer conversion for each polymerization sample was determined by 1H NMR in CDCl3 by comparing the shifts of the alkene protons of the monomer at 4.60 and 6.06 ppm to the growing polymer peaks at 4.21 and 4.09 ppm. The polymer was isolated by precipitation in diethyl ether to give pPAEMA. 1H NMR (400 MHz, DMSO-d6) δ: 7.79 (end group), 7.60 (end group), 7.44 (end group), 4.21, 4.09, 2.73, 2.12, 1.83−1.73, 1.45−0.77 ppm. Number-average molecular weight (Mn) in DMF = 15 700 Da; PDI = 1.25. Chain Extension of pPAEMA-RAFT with PEGMA. pPAEMA (160 mg, 1.01 × 10−5 mol, Mn 15 700, PDI 1.25), PEGMA (0.45 g, 9.9 × 10−4 mol), and AIBN (0.35 mg, 2.13 × 10−6 mol) were dissolved in DMF (1 mL). The solution was deoxygenated for 30 min by bubbling with argon and then immersed in a 70 °C bath. At 0.5, 1, 1.5, 2, and 3 h, samples were syringed from the incubating mixture under air-free

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RESULTS AND DISCUSSION The monomer with the required ketone moiety, PAEMA, was synthesized by esterification of levulinic acid with 2hydroxyethyl methacrylate (Scheme 1a). Polymerization of this monomer using ECPDB as the chain transfer agent (CTA) in DMF was initiated by thermolysis of AIBN at 70 °C (Scheme 1b). The kinetics of PAEMA polymerization are shown in Figure 1a. A linear relationship was observed between the semilogarithmic plot and time. In addition, the molecular weight increased linearly with monomer conversion up to 92%, with a corresponding decrease in PDI over the course of the reaction, consistent with a living polymerization. The distributions by GPC with increasing conversions are shown B

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Scheme 1. (a) Synthesis of Monomer and (b) Homopolymerization of PAEMA

Figure 2. DMF GPC traces of the evolution of molecular weight at different time intervals for the polymerization of PAEMA.

in Figure 2. The final polymer was purified by precipitation into diethyl ether, and the 1H NMR spectrum of the purified homopolymer is provided as Figure 3. The molecular weight of the final polymer was 15 700 Da, and the PDI was 1.25. Both the kinetic plots and low PDI suggest that this system is a controlled radical polymerization. As a result of RAFT polymerization with ECPDB, a dithioester was present at the ω-end of the polymer chain end. Although this moiety was not clearly visible in the spectrum taken in CDCl3, the chain end was clearly observed in DMSO-d6 (see Supporting Information, Figure S3) as resonances at 7.79, 7.60, and 7.44 ppm. Because the dithioester group remained intact after polymerization, the homopolymer could potentially be used as a macro-chain-transfer agent for chain extension reactions to form block copolymers. To investigate this, poly(ethylene glycol) methacrylate (PEGMA) was polymerized in the presence of pPAEMA, forming a block copolymer (Scheme 2). The polymerization conditions of [monomer]:[macroRAFT-CTA]:[initiator] of 465:5:1 resulted in the formation of pPAEMA-b-PEGMA. Similar to the behavior of the homopolymerization of PAEMA, the polymerization of PEGMA using pPAEMA as the macroRAFT-CTA resulted in a controlled radical polymerization up to 60% conversion (Figure S4b,c). GPC chromatograms of the initial PPAEMA and the subsequent block copolymer formation clearly showed increasing molecular weight with increasing reaction times (Figure S4a). A large shift of retention times between the two polymers was observed

Figure 3. 1H NMR spectrum (CDCl3) of purified pPAEMA.

and may be attributed to the dramatic change in the polarity of the copolymer after the addition of PEGMA fragments. As these hydrophilic fragments are added, the solubility of the macromolecule is expected to increase in polar media, resulting in an increase of the hydrodynamic volume as measured by GPC. After 60% conversion the PDI increased dramatically and the polymerization slowed significantly. The GPC trace of the polymer at 70% conversion was bimodal, and the molecular weight of the block copolymer was 55 000 Da with a PDI 1.6

Figure 1. RAFT polymerization of PAEMA. (a) Semilogarithmic kinetic plot and (b) evolution of molecular weight (MW) and PDI with respect to conversion. C

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Scheme 2. Chain Extension of PPAEMA-RAFT with PEGMEA

chains. The 1H NMR spectrum for the purified copolymer is depicted in Figure 4. As expected, the copolymer was significantly more water-soluble than pPAEMA due to the hydrophilic PEG side chains. A water-soluble copolymer is particularly useful for conjugating biomolecules via oxime coupling reactions for applications in the biological field. Next the reactivity of the ketone moiety was investigated. Given the ubiquity of hydrophobic drugs, BHA was first used as a model system to conjugate to pPAEMA (Scheme 3). Monitoring the conjugation by 1H NMR spectroscopy, within 3 h 100% of the ketone side chains reacted. Also, the signals of the phenyl group protons in CDCl3 shifted from 7.23 to 7.36 ppm, corresponding to the formation of the oxime bond. In addition, a shift of molecular weight by GPC was observed (Figure 5a). To isolate the functionalized polymer, the conjugate was purified by washing with an aqueous NH4Cl solution to remove trace excess of unreacted BHA and the triethylene amine hydrochloride salt generated from neutralizing the BHA. The 1 H NMR spectrum in deuterated DMSO of the polymer showed the presence of the benzyl group at 7.26 and 4.93 (Figure 5b). Comparison of the integration values of the protons in the conjugate revealed that all the ketone groups of the polymer reacted, confirming complete conjugation. In addition, the peak for the methyl protons adjacent to the ketone at 2.18 ppm was no longer visible. A peak from the dithioester group was still visible in the 1H NMR at 7.83 ppm; however, the integration value was reduced from the starting polymer, indicating that some cleavage had occurred. The conversion was quantitative, the reaction rate was relatively fast, and there appeared to be no side reactions. Thus, the oxime coupling reaction is comparable to that of the click coupling of azide and alkynes groups.12 The coupling was also confirmed by FTIR as shown in Figure 5c. A previously unobserved band

Figure 4. 1H NMR spectrum (DMSO-d6) of block copolymer PPAEMA-b-PPEGMA.

(Figure S4a). The shape of the curve indicated early termination and coupling, contributing to the larger molecular weight distribution. The homopolymer/macro-chain-transfer agent was formed at 92% conversion. Under these conditions some of the RAFT end groups can be lost. Efficient block formation has been often observed for RAFT polymers, especially if the original block was isolated from polymers taken to a limited conversion.54 The polymer stopped at 60% conversion was isolated and had a Mn of 30 000 Da and a PDI of 1.32. Given the unique polarities of the side chains, molecular weights were determined by comparing protons of the pPAEMA and pPEGMA side Scheme 3. Coupling of O-Benzylhydroxyamine to pPAEMA

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Figure 6. 1H NMR (CDCl3) of pPAEMA with tetra(ethylene glycol) side chains.

Figure 7. Mouse fibroblasts were subjected to 0.01, 0.1, and 1.0 mg/ mL of pPAEMA−tetra(ethylene glycol) for 24 h before undertaking a MTT assay. The results are shown as percent viability compared to the control with media only. Each sample was replicated 5 times and the control with no polymer 10 times. Student’s t test revealed no statistical difference between the concentrations.

at 1629 cm−1 in the starting polymer appeared in the functionalized macromolecule, corresponding to the CN absorption band of the oxime bond. This resonance is typically weak for oxime bonds. In addition, the CO absorption of the conjugate was shifted from 1719 cm−1 of the pPAEMA to 1731 cm−1 of the modified polymer. The decrease in the intensity and the peak shift may be due to the absence of the ketone groups in the conjugate compared to the homopolymer.

Figure 5. Coupling of BHA to the homopolymers. (a) THF GPC monitoring of the MW increase after coupling of BHA. (b) 1H NMR (DMSO-d6) of polymer after conjugation and purification. (c) FTIR analysis of the oxime conjugation.

Scheme 4. Coupling of Carboxymethoxyamine to Ketone Homopolymer

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To demonstrate the versatility of the conjugation, carboxymethoxyamine (CMA), a hydrophilic aminooxy compound, was also conjugated to pPAEMA (Scheme 4). This small molecule is an amino acid mimic and addition of the compound to N-termini of peptides enables chemoselective attachment to form oxime chemistry without the need for protection of the side chains.36 Using a mixed media of CDCl3 and CH3OH (1:1) to solubilize the reagents, the conjugation of CMA to pPAEMA was also found to be efficient and completed within 3 h. As for BHA, the molecular weight increased after conjugation (Figure S5a). The 1H NMR spectrum of the polymer showed an extra peak at 4.5 ppm corresponding to the methylene of CMA (Figure S5b). Again, the peak at 2.18 corresponding to the ketone was not visible, indicating complete conjugation. The dithioester group was still visible. The addition of the carboxylic acid moiety on the side chains increased the polymer hydrophilicity significantly so that the polymer was soluble in water. Oligo(ethylene glycol) side chain units were introduced into the polymer side chains in order to increase the biocompatibility of the polymer. O-Hydroxylamine tetra(ethylene glycol) was incubated with pPAEMA in the presence of sodium acetate. As for BHA and CMA, the conjugation was efficient. Peaks corresponding to the tetra(ethylene glycol) were observed at 3.65−3.38 ppm (Figure 6). A minor peak was observed corresponding to residual ketone. The dithioester group was no longer visible, potentially due to the increased time used to conjugate the TEG in this case. The cytotoxicity of this polymer was then determined for mouse fibroblast cells. The cells were incubated with increasing concentrations of polymer 0.01, 0.1, and 1 mg/mL for 24 h before undertaking the MTT assay. The conjugate was found to be noncytotoxic at all concentrations tested for this cell line as no loss in cell viability was observed (Figure 7). This data indicates that this approach to synthesize polymers may result in biocompatible materials, applicable in medicine. It can be envisioned that many different small molecules, drugs, and peptides can be readily conjugated to this versatile and stable polymer backbone utilizing the efficient oxime click reaction.55

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of monomer, polymers not in text, kinetic evaluation of block polymerization, and CMA conjugate data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (H.D.M.); t.davis@unsw. edu.au (T.P.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the NIH (R21 CA 137506-01) and the Australian Research Council (ARC Discovery Project Grant, DP 0770818) for funding the research. G.J.S. thanks the UC Nanotoxicology Research and Training Program for a fellowship. Dr. Erhan Bat is thanked for help with the NMR figures.

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REFERENCES

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CONCLUSION A ketone-containing monomer, PAEMA, was synthesized and polymerized to form homo- and block copolymers by RAFT polymerization. The synthesis of the homopolymer was found to be well-controlled and yielded a low dispersity polymer. The macroRAFT CTA resulted in controlled polymerization of PEGMA up to 60% conversion, after which bimodal molecular weight distributions were observed. The resulting block copolymer, pPAEMA-b-PEGMA, was soluble in water. OHydroxylamine compounds were readily attached to the polymer backbone with high efficiency. Both hydrophobic (BHA) and hydrophilic moieties (CMA and tetra(ethylene glycol)) were conjugated. Initial cytotoxicity work with mouse fibroblast cells revealed that an oligo(ethylene glycol)substituted pPAEMA was noncytotoxic up to at least 1 mg/ mL. This work demonstrated that the oxime covalent coupling to this keto polymer can be a very useful methodology to prepare conjugates. Future work will focus on the utilization of the ketone bearing polymers to form drug and peptide− polymer conjugates for therapeutic delivery and personalized medicine applications. F

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dx.doi.org/10.1021/ma302183g | Macromolecules XXXX, XXX, XXX−XXX